Development and function of the voltage-gated sodium current in immature mammalian cochlear inner hair cells.

Department of Biomedical Science, University of Sheffield, Sheffield, United Kingdom.

Abstract

Inner hair cells (IHCs), the primary sensory receptors of the mammalian cochlea, fire spontaneous Ca(2+) action potentials before the onset of hearing. Although this firing activity is mainly sustained by a depolarizing L-type (Ca(V)1.3) Ca(2+) current (I(Ca)), IHCs also transiently express a large Na(+) current (I(Na)). We aimed to investigate the specific contribution of I(Na) to the action potentials, the nature of the channels carrying the current and whether the biophysical properties of I(Na) differ between low- and high-frequency IHCs. We show that I(Na) is highly temperature-dependent and activates at around -60 mV, close to the action potential threshold. Its size was larger in apical than in basal IHCs and between 5% and 20% should be available at around the resting membrane potential (-55 mV/-60 mV). However, in vivo the availability of I(Na) could potentially increase to >60% during inhibitory postsynaptic potential activity, which transiently hyperpolarize IHCs down to as far as -70 mV. When IHCs were held at -60 mV and I(Na) elicited using a simulated action potential as a voltage command, we found that I(Na) contributed to the subthreshold depolarization and upstroke of an action potential. We also found that I(Na) is likely to be carried by the TTX-sensitive channel subunits Na(V)1.1 and Na(V)1.6 in both apical and basal IHCs. The results provide insight into how the biophysical properties of I(Na) in mammalian cochlear IHCs could contribute to the spontaneous physiological activity during cochlear maturation in vivo.

a, Voltage responses from an apical-coil and a basal-coil P4 IHC with 1.3 mM Ca2+ in the extracellular solution. Note the more irregular spontaneous action potential activity in apical IHCs, in agreement with previous observations . Cell properties were, apical: Vm −55 mV, Cm 8.9 pF, Rs 3.3 MΩ, gleak 4.7 nS; basal: Vm −54 mV, Cm 8.3 pF, Rs 1.9 MΩ, gleak 4.3 nS. b, Percentage of apical and basal IHCs found to be spontaneously active at the different postnatal age tested (P2–P12). Note that under the above recording conditions, no IHCs were found spontaneously active during the second postnatal week. Number of IHCs tested is shown above each bar of the histogram. c, Spontaneous action potentials are reversibly abolished during the superfusion of a nominally Ca2+-free solution. Apical P3 IHC: Vm −59 mV. Unless otherwise stated all recordings in this and following Figures are near body temperature (34–37°C).

Inward currents (INa+ICa: black traces) recorded from a P2 apical IHC obtained in the presence of K+ channel blockers. Recordings were obtained by applying a depolarizing voltage step to −17 mV from −110 mV. In this and the following figures actual test potentials, corrected for voltage drop across uncompensated Rs, are shown next to the traces. Red traces show the isolated INa obtained by subtracting the current in the presence of NMDG+ (a: grey trace) or TTX (b: grey trace) from total inward current (black traces). The small residual current in NMDG+ or TTX (1 µM: grey traces) is the isolated ICa. Cm 7.7 pF, Rs 0.7 MΩ, gleak 1.6 nS.

a, A spontaneous action potential (AP: top trace) was used as a voltage command to elicit INa (bottom trace) from the holding potential of −60 mV in four P2–P3 apical IHCs. The AP was recorded from an immature IHC under current clamp conditions at body temperature. b, Spontaneous action potential activity under current clamp recorded from a P2 basal IHC. The arrow indicates a spontaneous IPSP due to currents flowing through ACh receptors.

Apical (a–c, g–i) and basal (d–f, j–l) cochlear turns of immature rat (P5) immunolabeled with antibodies against NaV 1.1 (a, d; red) and NaV 1.6 (g, j; red). Note that both IHCs and outer hair cells (OHCs) are labeled. Anti-NaV 1.1 antibody labeling was mainly found in the supranuclear region of IHCs (arrow) and OHCs (arrowheads) and less intensely present in the cell membrane, which could reflect a high rate of channel turnover. Labelling was absent in adult IHCs (P20: data not shown), which do not have a Na+ current. Labeling for NaV 1.6 was more obviously located to the membrane in both cell types. Co-labeling with anti-neurofilament antibody (NF200, green) showed no expression of NaV 1.1 or NaV 1.6 in afferent nerve fibres. Scale bars represent 10 µm. Cell nuclei are marked by DAPI (blue).